CD1d transmembrane glycoproteins present lipids to Natural Killer T (NKT) cells and activate them to produce cytokines critical for initiation of immune T cell responses. The mechanisms responsible for lipid association are incompletely understood, but lipid binding can occur in the endoplasmic reticulum (ER), the secretory pathway and the endocytic pathway. We seek to understand the mechanisms that regulate assembly of the human CD1d glycoprotein in the ER with its small subunit 22m and with ER-derived lipids. We also wish to determine the mechanisms responsible for exchanging associated lipids during secretion and during recycling through the endocytic pathway. We also will investigate the mechanism used by herpes simplex virus-1 (HSV-1) that allows cells infected by it to evade recognition by NKT cells. This involves the specific redistribution of human CD1d molecules from the cell surface to the limiting membrane of the lysosome. CD1d molecules associate with MHC class II molecules, which are regulated by expression of the class II transcriptional activator CIITA. CD1d redistribution upon HSV-1 infection is not observed in transfected cell lines that co-express CIITA, but is seen in HSV-1-infected class II-positive dendritic cells, suggesting that in dendritic cells the CD1d-MHC class II linkage is uncoupled upon infection. We propose to determine the mechanism underlying this phenomenon. Dendritic cells, macrophages, and other cell types are activated by the interaction of a variety of pathogen components with innate immune receptors such as Toll-like Receptors (TLRs). One outcome of such activation is that the activated cells robustly stimulate NKT cells, in part because of a change in the profile of CD1d-associated lipids. Macrophages undergo a reprogramming of their lysosomal targeting mechanisms upon TLR4-mediated activation that results in the secretion of precursors of lysosomal proteins. These proteins include lysosomal enzymes and prosaposin, the precursor of the lipid transfer proteins called saposins. One of these, saposin B, mediates lipid exchange by CD1d molecules in the endocytic pathway. We will determine the role of this reprogramming in the enhancement of NKT stimulation seen upon TLR-mediated activation. We also propose to generate a mouse strain in which the mouse CD1d locus is replaced by the human one, to allow us to evaluate the in vivo role of CD1d down regulation in the infectious process. PUBLIC HEALTH RELEVANCE: CD1d molecules play a vital role in initiating the appropriate T cell responses to many viruses by activating NKT cells. Understanding how they mediate this function is of vital importance. Herpes simplex virus is a major public health problem and its ability to avoid NKT recognition is likely to be key for its persistence after the initial infection.